The invention relates to methods for medicinal diagnosis and to substances for obtaining such diagnoses. More specifically, the substances are visualization polymers of molecules such as proteins, enzymes, and chemically tagged polyols, polyolefins, carbohydrates and natural or synthetic polypeptides, which can be combined with biological material and provide substantial chemical amplification of the quantity of material detected.
The requirements for a medical diagnostic method, which detects and/or quantifies the presence of biological material, include identification of extremely small quantities and selection of a single species of material from a complex mixture containing similar species. In the past, such methods as radiolabeling, radiobioassay and immunoassay techniques have formed the basis for such diagnostic medicine. For example, immunological reagents have been used extensively for detecting and/or quantitating a broad spectrum of molecular species such as proteins, lipids, carbohydrates, steroids, nucleic acids, drugs, carcinogens, antibiotics, inorganic salts etc. Indeed, polyvalent and monoclonal antibodies are very important diagnostic tools in most areas of clinical medicine today.
During the past 40 years, a variety of procedures have been developed to visualize specific antigen-antibody interactions fluorimetrically or colorimetrically. Since the utility of immunodiagnostic procedures often depends upon the sensitivity and the specificity with which the target antigen or molecule can be detected, new methods for increasing these detection parameters are highly desirable. The scientific and patent literature is replete with work designed with this goal in mind. A detailed discussion of the advantages and disadvantages of immunologic methods can be found in any standard textbook on immunocytochemistry; see for example, L. A. Sternberger, "Immunohistochemistry", 2nd Ed., John Wiley and Sons, New York, 1979.
Immunologic detection methods can utilize direct or indirect visualization techniques for measurement of the formed immune complex. In general, these methods visually indicate the presence of the complex through use of an entity coupled to the complex which produces a detectable, quantifiable signal such as color, fluorescence, radioactivity, enzymatic action and the like. The more signal intensity present per complex, the better will be the sensitivity for the presence of a minute quantity of target molecule.
Of these methods, the simplest and least sensitive is direct immunofluorescence. In this method, a primary antibody (or specific ligand-binding protein) is chemically linked to a fluorochrome, such as rhodamine or fluorescein which functions as the signal entity.
Indirect immunofluorescence methods, in which a primary antibody is used unmodified and it, in turn, is detected with a fluorescently-labeled secondary antibody, generally will increase the detection sensitivity about two to four-fold over direct methods. An additional three to five-fold enhancement in sensitivity has been reported using a "haptene-antibody sandwich" technique; see Cammisuli, et al., J. Immunol., 117,1695 (1976); Wallace, et al., J. Immunol Methods, 25, 283 (1979). According to this technique, ten to fifteen molecules of a small haptene determinant such as 2,4-dinitrophenol are chemically coupled to each primary antibody molecule. Then, by use of a fluorescently-labeled second antibody which complexes with the haptene molecules, rather than with the primary antibody itself, more of the secondary visualization protein can be bound per antigen site, thus further increasing the sensitivity.
Nakane and associates; see Nakane, et. al., J. Histochem. Cytochem., 22, 1084 (1974), Wilson, et. al. "Immunofluorescence and Related Staining Techniques", W. Knapp, H. Holuban and G. Wick, Eds. Elsevier/North-Holland Biomedical Press, p. 215; have coupled secondary antibodies to monomeric horseradish peroxidase and used the catalytic activity of peroxidase enzyme to reveal either the site, or the amount, of antigen in the test sample. Similar enzymatic assays have been developed with intestinal or bacterial alkaline phosphatase conjugated secondary antibodies; see Avrameas, Immunochemistry, 6, 43, (1969); Mason, et. al., J. Clin. Path., 31, 454 (1978).
The enzymatic signal of this method may occur in either of two ways. Enzymatic conversion of a soluble enzyme substrate into an insoluble, colored product permits the direct localization of the antigen by direct macroscopic visualization or by light microscopic examination. Alternatively, colorless substrates can be enzymatically converted into soluble colored products which can be used to quantitate antigen concentrations by direct colorimetric analysis. The latter method is the basis of the Enzyme-Linked Immuno-Sorbent Assay (ELISA), which is widely used in clinical laboratories around the world; see Sternberger, Immunohistochemistry, 2nd Edition, John Wiley and Sons, N.Y. (1979); Engvall, et. al., Immunochem., 8, 871 (1972); Engvall, et al., J. Immunol., 109, 129 (1972); Guesdon, et. al., J. Histochem. and Cytochem., 27, 1131 (1979); Voller et. al., "The Enzyme Linked Immuno Sorbent Assay (ELISA)", Dynatech Laboratories Inc., Alexandria (1979).
These enzyme-based detection methods are generally more sensitive than direct or indirect immunofluorescence methods since the high turnover of substrate by the enzyme continuously accumulates a measurable product over long periods of time.
To further increase the sensitivity of immunoenzyme assays, Sternberger; see Sternberger, et. al. J. Histochem, Cytochem. 18, 315 (1970) developed a three stage peroxidase-antiperoxidase (PAP) assay method. Following the addition of a primary antibody and a secondary antibody, which acts as a bridge between the primary antibody and antiperoxidase antibody, a peroxidase-antiperoxidase antibody complex (PAP complex) is added to the sample prior to the development of the enzymatic reaction. Since the PAP complex contains two immunoglobulins (antiperoxidase antibodies) and three active peroxidase molecules, the net effect is to provide more enzyme at the antigen site with which to amplify the detection signal. Although quite useful, the PAP detection system has limitations. The secondary "bridge" antibody must be used at saturating levels to ensure optimal binding of the PAP complex. Furthermore, the antiperoxidase and the primary antibody must be of the same, or an immunologically cross-reacting, species so that the secondary antibody will bridge to both.
During the past few years it has been shown that the specific and tenacious interaction between biotin, a small water soluble vitamin, and avidin, a 68,000 dalton glycoprotein from egg white, can be exploited to develop antigen or ligand detection systems, see Bayer and Wilchek in Voller, et. al., "The Enzyme Linked Immuno Sorbent Assay (ELISA)", Dynatech Laboratories Inc., Alexandria (1979). Biotin can be covalently coupled to amino, carboxyl, thiol or hydroxyl groups present in proteins, glycoproteins, polysaccharides, steroids and glycolipids using well established chemical reactions; see Guesdon, et. al., J. Histochem. and Cytochem., 27, 1131 (1979); Sternberger, et. al., J. Histochem. Cytochem., 18, 315 (1970); Bayer, et. al., Methods Biochem. Anal., 26, 1, (1980); Bayer, et. al., J. Histochem. Cytochem., 24, 933 (1976); Heitzmann, et. al., Proc. Natl. Acad. Sci. USA, 71, 3537 (1974). Biotin can also be introduced into other macromolecules, such as DNA, RNA and co-enzymes, by enzymatic methods that utilize biotin-labeled nucleotide precursors; see Langer, et al., Proc. Natl. Acad. Sci. USA, 78, 6633 (1981). Similarly, avidin can be coupled to a host of molecular species by standard chemical reactions; see Sternberger, "Immunohistochemistry", 2nd Edition, John Wiley and Sons, N.Y. (1979); Nakane, et. al., J. Histochem. Cytochem., 22, 1084 (1974); Guesdon, et. al., Histochem. and Cytochem., 27, 1131 (1979); Bayer et. al., Methods Biochem. Anal., 26, 1, (1980). This allows for great flexability in designing detection systems for use in immunology, immunopathology and molecular biology.
In 1981 Hsu; see Hsu, et. al., Amer. J. Clin. Path., 75, 734 (1981); Hsu, et al., J. Histochem. Cytochem., 29, 577 (1981); reported the use of avidin-biotinylated horseradish peroxidase complex (ABC) for antigen detection. In their three-step procedure, the primary antibody incubation is followed by an incubation period with a biotin-labeled secondary antibody and then with the ABC complex, formed by preincubating avidin with a titrated amount of biotinylated peroxidase. Since avidin has four biotin-binding sites per molecule, at least three peroxidase enzymes can be added to avidin without interfering with its ability to interact with the biotinylated secondary antibody. Hsu and associates; see Hsu et. al., Amer. J. Clin. Path., 75, 734 (1981); Hsu, et. al., J. Histochem. Cytochem., 29, 577 (1981); reported that the ABC detection procedure was 4-8 times more sensitive in detecting antigens in tissues than either the immunoperoxidase or the PAP detection systems. Madri; see Madri, et. al., Lab. Invest., 48, 98 (1983); has confirmed these observations and shown that the ABC method is four-fold more sensitive for antigen detection using an ELISA system than either the immunoperoxidase or the PAP techniques. By all criteria tested, the ABC method is the most sensitive detection procedure used in clinical diagnostic labs to date.
The limit of sensitivity for the ABC method, however, appears to be 30 to 100 pg of a target molecule such as a protein or nucleic acid. This is significantly higher than the upper limit required for detection of a single molecule per cell. Limits for other less sensitive methods are even higher. Accordingly, it is an object of the invention to develop visualization methods which substantially improve sensitivity over that provided by known visualization techniques. Yet another object is development of a stable, easily manipulated visualization system which has a long shelf life. Finally, as with any diagnostic technology, an ultimate goal would be development of a capacity for detecting a single molecule of a species in any given cell.